GLP-1 (glucagon-like peptide-1) is a peptide of intestinal origin that is deeply involved in regulation of glucose homeostasis. GLP-1 is synthesized in intestinal L-cells by the tissue-specific post-translational processing of preproglucagon which is a glucagon precursor and released into circulation in response to food intake. This peptide serves as main mediators of the enteroinsular axis and act through the binding to particular receptors.
GLP-1 has been known to act mainly on the pancreas and promote the insulin release of β cells in a glucose concentration-dependent manner. It has also been suggested that GLP-1 is likely to suppress glucagon secretion, delay gastric emptying, and enhance peripheral glucose disposal.
The administration of GLP-1 to patients with non-insulin-dependent diabetes mellitus can normalize postprandial glucose levels, suggesting that GLP-1 may be used as a therapeutic drug. GLP-1 also has the effect of improving glycemic control in patients with insulin-dependent diabetes mellitus. Since the effect of promoting insulin release by GLP-1 depends on plasma glucose concentrations, GLP-1 mediates reduced insulin release at a low plasma glucose concentration and therefore advantageously causes no serious hypoglycemia. Thus, the highly safe treatment of diabetes can be achieved by controlling the amount of GLP-1 in blood as necessary. However, the half-life of GLP-1 in blood is as extremely short as 2 to 6 minutes, presenting the problem of its limited possibility as a therapeutic agent.
To solve such a problem, an attempt has been made to modify GLP-1. For example, Patent Document 1 discloses a PEGylated GLP-1 compound comprising a GLP-1 compound conjugated to at least 1 polyethylene glycol (PEG) molecule. In the PEGylated GLP-1 compound, each PEG is bound with the GLP-1 compound at the Cys or Lys amino acid or at the carboxyl-terminal amino acid. The PEGylated GLP-1 compound has an elimination half-life of at least 1 hour.
According to Patent Document 1, the obtained biologically active peptide has a longer half-life and highly delayed clearance compared to those of unPEGylated peptides. It has also been shown that the PEGylated GLP-1 compound and composition are useful in the treatment of the health condition such as diabetes, obesity and irritable bowel syndrome as well as reducing blood sugar level, suppressing gastric and/or intestinal motility, gastric and/or intestinal emptying, and controlling food intake (e.g., Non-patent document 1).
However, PEG is a compound that is not metabolized in vivo. Therefore, the continuous administration of the PEGylated GLP-1 compound accumulates PEG in vivo and might cause adverse reaction in the living bodies (Non-patent document 1).
Moreover, to prolong the half-life, a method for adding an oligosaccharide chain to GLP-1 or modified GLP-1 has also been proposed (e.g., Patent Documents 3 and 4). Patent Document 3 discloses a method which comprises introducing an oligosaccharide chain added amino acid to positions 26, 34 and/or 37 of GLP-1, etc. However, the type of the oligosaccharide chain and the oligosaccharide chain added sites are less than optimal. On the other hand, Patent Document 4 discloses a method which comprises binding modified hyaluronic acid having a molecular weight of about 200 KDa to a GLP-1 analog. However, when such big hyaluronic acid molecules are produced in large amounts, it is difficult to make their lengths or structures uniform. Thus, the actual hyaluronic acids may largely vary in structure or length. Oligosaccharide chain added peptides of uniform length or structure are required for pharmaceutical use.
Exendin-4 found from the saliva of a lizard (Heloderma) is a compound that is structurally similar to GLP-1 and has similar activity and high stability in blood (Non-patent Document 2) which has been placed on the market in U.S. However, exendin-4 has a nonhuman sequence and might induce neutralizing antibodies attributed to long-term administration, leading to attenuated efficacy ((Non-patent Documents 3-5).
On the other hand, it has become evident that oligosaccharide chains play various roles in vivo. They have been less well studied due to their complicated and diverse structures, though the importance of the studies is recognized. An attempt has been made on a method for obtaining a glycopeptide having constant composition (Patent Document 2). However, this production method is still less than sufficient from the viewpoint of convenience or large-scale production and is not practical method particularly for long oligosaccharide chains existing in vivo.    [Patent Document 1] National Publication of International Patent Application No. 2006-520818    [Patent Document 2] WO 2005-095331    [Non-patent document 1] Toxicological Science, 42, 152-157 (1998)    [Non-patent document 2] J Biol. Chem. 267, 7402-5 (1992)    [Non-patent Document 3] Vascular Health and Risk Management 2, 69-77 (2006)    [Non-patent Document 4] JAMA. 298, 194-206 (2007)    [Non-patent Document 5] Endocrine Reviews 28, 187-218 (2007)